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Oceanological and Hydrobiological Studies Oceanological and Hydrobiological Studies International Journal of Oceanography and Hydrobiology Volume 44, Issue 1, March 2015 ISSN 1730-413X pages (68-73) eISSN 1897-3191 Contracaecum spp. from endemic Baikal fishes: the Baikal yellowfin Cottocomephorus grewingkii (Dybowski, 1874) and the longfin Baikal sculpin Cottocomephorus inermis (Yakovlev, 1890) by Abstract 1 Olga Rusinek All the nematodes found in body cavities of the examined 2 endemic Baikal fishes: 88 Baikal yellowfin Cottocomephorus Marek Kulikowski grewingkii (Dybowski, 1874) and 35 longfin Baikal sculpin Katarzyna Najda2 Cottocomephorus inermis (Yakovlev, 1890) were identified as 2,* Contracaecum osculatum baicalensis (Mozgovoi and Ryjikov, Jerzy Rokicki 1950) L3 larvae. The prevalence, mean intensity, intensity range and abundance of the nematodes in C. grewingkii were 37.5%, 2.55, 1-31, and 0.96, respectively, the corresponding values in C. inermis were 60.0%, 2.43, 1-10, and 1.46. The infestation level in C. grewingkii was significantly higher than DOI: 10.1515/ohs-2015-0007 in C. inermis (Mann-Whitney U-test, p<0.02). The number of parasites was found to increase with the fish length. Although Category: Original research paper in both and C. inermis, the anisakids were more frequent Received: September 29, 2014 in males (prevalence of 52.17 and 67.76%, respectively) than Accepted: November 19, 2014 in females (prevalence of 35.39 and 42.86%, respectively), differences between the sexes in the infestation level in the two species were not significant (Mann-Whitney U-test, P= 0.09 and P=0.23, respectively). The molecular method applied (PCF-RFLP) allows to identify all the nematodes in 1 Baikal Museum of Irkutsk Scientific Center both examined fish species as C. osculatum baicalensis. of Siberian Branch of the Russian Academy of Sciences, Akademicheskaya str.,1, 664520 Listvyanka, Russia 2Department of Invertebrate Zoology and Parasitology, University of Gdańsk, ul. Wita Stwosza 59, 80-308 Gdańsk, Poland Key words: Contracaecum osculatum baicalensis, Anisakidae, Cottocomephorus, PCR - RFLP * Corresponding author: [email protected] The Oceanological and Hydrobiological Studies is online at oandhs.ocean.ug.edu.pl ©Faculty of Oceanography and Geography, University of Gdańsk, Poland. All rights reserved. Oceanological and Hydrobiological Studies, VOL. 44, ISSUE 1 | MARCH 2015 69 Contracaecum osculatum baicalensis from Baikal Cottocomephorus spp. Mattiucci & Nascetti 2008). Molecular data show C. Introduction osculatum baicalensis to be genetically different from the remaining five C. osculatum sibling species and The parasitic nematode Contracaecum osculatum validate its identification as a true biological species baicalensis (Mozgovoi and Ryjikov 1950) of the within the C. osculatum complex (D’Amelio et al. family Anisakidae is a common endemic species 1995). At the larval stage, C. osculatum baicalensis in Lake Baikal (Rusinek 2007). The nematode’s was recognized by allozymes in the Baikal endemic complex life cycle involves at least two intermediate fish species: C. grewingkii, C. inermis, Comephorus hosts (Sudarikov & Ryzhikov 1951). Hypothetically, baicalensis, Coregonus lavaretus, C. autumnalis some invertebrate organisms: the dominant Baikal migratorius and Thymallus arcticus, which represent zooplankter Macrohectopus branickii (Dybowsky prey items of Pusa sibirica in Lake Baikal (Mattiucci 1874) (Amphipoda, Gammaridae) and harpacticoid & Nascetti 2008). copepods may serve as the first intermediate hosts We hypothesize that new information regarding (Bauer 1987). It is reasonable to assume that the first the Baikal Cottocomephorus spp. parasites is essential intermediate hosts occur in abundance (Rusinek for exploring changes in the infection level and 2007). The second intermediate hosts include fish for refining the identification techniques. This species endemic to Lake Baikal: the benthopelagic information will allow us to identify the distribution Baikal Cottocomephorus grewingkii (Dybowski 1874) of intermediate hosts in Lake Baikal and to arrive at and the longfin Baikal sculpin, Cottocomephorus a better understanding of host-parasite interactions. inermis (Yakovlev 1890) (Scorpaeniformes, Cottoidei), which support the parasites as L3 larvae in their coelom (Lâjman 1933, Dogel’ et al. 1949, Materials and methods Sudarikov & Ryzhikov 1951, Zaika 1965, Rusinek et al. 2007). In addition, many other fish species Parasites (salmonids, coregonids, thymallids, and lotids) can act as the second intermediate hosts of C. The material for this study was collected from 88 osculatum baicalensis (Zaika 1965, Rusinek 2007). and 35 individuals of C. grewingkii and C. inermis, The nematode reaches the maturity in the stomach respectively, caught in July-September 2012 near and intestine of the Baikal seal Pusa sibirica (Gmelin Listvyanka, on the southern shore of Lake Baikal 1788) which feeds on the fish species mentioned (51°51’ 05.00”N; 104° 51’ 55.00”E). Each fish was above. The Baikal seal is the only known definitive sexed and measured (lt) to the nearest 1 mm. The host of the parasite (Sudarikov & Ryzhikov 1951, C. grewingkii specimens were divided into 3 length Rusinek 2007). classes, while C. inermis into 2 classes – on account As in other anisakid species complexes e.g. of their length distribution – so that the number of Anisakis simplex (Mattiucci et al. 1997), speciation fish in each interval was more than 15. Nematodes within the C. osculatum complex appears to be were isolated from host body cavities, rinsed in accompanied by poor morphological differentiation, water, and stored in 70% ethanol until further and morphological identification of sibling species is analysis. Subsequently, each parasite was dissected unreliable at both larval and adult stages. In contrast, into 3 parts. Semi-permanent mounts were prepared nematode identification at any developmental stage is of the anterior and posterior part of each specimen possible by molecular methods (Kijewska et al. 2002), (Rolbiecki 2002) and examined under a compound which is important for the study of the parasites’ life light microscope. The nematodes were identified cycles, biology, taxonomy, and pathology they may using the identification key (Bauer 1987). The middle cause. These anisakid taxa can be characterized part of each individual was fixed in 70% ethanol and using several different molecular markers, including used for molecular identification. allozymes, nuclear ribosomal DNA (rDNA), internal transcribed spacer regions (ITS), single strand DNA isolation and amplification conformation polymorphism mitochondrial DNA (mtDNA), and cytochrome oxidase 2 (cox2) sequence The morphological identification of nematodes analyses (Zhu et al. 1998, Mattiucci & Nascetti 2007, was checked on randomly selected specimens ©Faculty of Oceanography and Geography, University of Gdańsk, Poland. All rights reserved. 70 Oceanological and Hydrobiological Studies, VOL. 44, ISSUE 1 | MARCH 2015 Olga Rusinek, Marek Kulikowski, Katarzyna Najda, Jerzy Rokicki digested by endonucleases, using a molecular (1982). All the statistical analyses were performed key based upon DNA patterns of the ITS1-5.8S- with STATISTICA 12.0. Non-parametric tests ITS2 fragment (Zhu et al. 2000, Dzido 2011). The (Mann-Whitney U-test, and Spearman’s rank nematode DNA was isolated as described by Hoarau correlation) were used because the distribution of et al. (2002). Amplification of an rDNA fragment was parasites deviated from the normal distribution due accomplished according to Zhu et al. (1998). Each to the accumulation of larvae and diet preferences of reaction mixture (total volume of 16 µl) contained the fish. The two fish species were compared in terms 1 µl of isolated genomic DNA, 1 U DyNAzyme II of their nematode infection level. The frequency DNA Polymerase (Finnzymes, Vantaa, Finland), distributions of parasite-fish sex correlation was dNTPs (250 µM each), 100 µM of each primer [NC2 examined as well. In addition, the data were tested and NC5 (Zhu et al. 1998)] and buffer 1 × (10 mM for significance of differences in the load of parasites Tris-HCl (pH = 8.4), 50 mM KCl, 0.1% Triton X-100, between the length classes. and 1.5 mM MgCl2). Amplification was carried out in a Techne Progene (Stone, U.K.) thermocycler Results as follows: initial denaturation for 5 min. at 94°C, followed by 30 cycles for 30 sec. at 94°C, annealing Morphological identification resulted in assigning at 60°C for 30 sec., and extension at 72°C for 30 sec., all the nematodes isolated from the yellowfin and terminated by the extension cycle at 72°C for 5 min. longfin Baikal sculpin to Contracaecum osculatum PCR products were separated electrophoretically baicalensis (Mozgovoi & Ryjikov 1950) L3 larvae. on 1% agarose gels and visualized by staining with In C. grewingkii, the infestation prevalence, mean ethidium bromide. intensity, intensity range, and nematode abundance Restriction fragment length polymorphism were 37.5%, 2.55, 1-31, and 0.96, respectively The amplified DNA samples were digested with (Table 1). When assessed for the yellowfin sex- TaqI, RsaI, XbaI, and BsuRI (Fermentas, Vilnius, related distribution (65 females and 23 males), the Lithuania) restriction enzymes and the products anisakids proved to occur more frequently in males were separated electrophoretically on 4% agarose gels than in females (prevalence of 52.17 and 35.39%, and visualized by staining with ethidium bromide respectively), but they were more numerous females (Sambrook
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